A conserved upstream motif orchestrates autonomous, germline-enriched expression of Caenorhabditis elegans piRNAs

Abstract

Piwi-interacting RNAs (piRNAs) fulfill a critical, conserved role in defending the genome against foreign genetic elements. In many organisms, piRNAs appear to be derived from processing of a long, polycistronic RNA precursor. Here, we establish that each Caenorhabditis elegans piRNA represents a tiny, autonomous transcriptional unit. Remarkably, the minimal C. elegans piRNA cassette requires only a 21 nucleotide (nt) piRNA sequence and an ∼50 nt upstream motif with limited genomic context for expression. Combining computational analyses with a novel, in vivo transgenic system, we demonstrate that this upstream motif is necessary for independent expression of a germline-enriched, Piwi-dependent piRNA. We further show that a single nucleotide position within this motif directs differential germline enrichment. Accordingly, over 70% of C. elegans piRNAs are selectively expressed in male or female germline, and comparison of the genes they target suggests that these two populations have evolved independently. Together, our results indicate that C. elegans piRNA upstream motifs act as independent promoters to specify which sequences are expressed as piRNAs, how abundantly they are expressed, and in what germline. As the genome encodes well over 15,000 unique piRNA sequences, our study reveals that the number of transcriptional units encoding piRNAs rivals the number of mRNA coding genes in the C. elegans genome.

Figure 1. Over 70% of 21U RNAs show distinct germline enrichment.

(A) Pipeline for computational identification of male and female 21U RNAs. A majority of 21U RNAs are classified as male or female germline-enriched. Pie chart depicts classification as proportion of 13,711 21U RNAs analyzed. (B,C) Male 21U RNAs are more highly expressed in male animals, and female 21U RNAs are more highly expressed in female animals. Relative expression of representative 21U RNAs was assayed by Taqman RT-qPCR in him-8(e1489) (B) and fog-2(q71) (C) male versus fem-1(hc17) female animals and normalized to non-enriched 21U RNA 21UR-1. Error bars: ±1 standard deviation (SD) of two biological replicates. AU: arbitrary units.

Figure 2. Variation in the core upstream motif correlates with 21U RNA germline enrichment.

(A) Spacer lengths follow expected distribution for all enrichment classifications. Dotted lines: canonical spacer length range (35–42 nt). (B) Male, but not female, 21U RNA loci show enrichment for core motifs with 5′ cytidines. Significantly fewer female 21U RNAs exhibit a GTTTC-containing core motif than male. Top: Weblogo plots illustrate core motif differences. Bottom: Pie charts depict proportions of 21U RNAs with GTTTC-containing core motifs indicating the 5′ nt (colors) or with no GTTTC-containing core motif (NM, no motif, dark grey). (C) Core motif variations correlate with male 21U RNA abundance in 5′-monophosphate-dependent libraries. Average 21U RNA abundance was calculated based on the 5′ nt of the core motif. Error bars: ±1 standard error of the mean (SEM). (D) Core motif variations do not correlate with female 21U RNA abundance in 5′-monophosphate-dependent libraries. Average 21U RNA abundance was calculated as in (C).

Figure 3. A transgenic synthetic 21U RNA shows characteristics of endogenous 21U RNAs.

(A) Diagram of Tg (dark grey) and Min (light grey) transgenes with core motif sequences shown. Asterisk indicates a 21U RNA whose core motif is disrupted by 21UR-synth and is therefore predicted not to express. (B) 21UR-synth is methylated by HENN-1. 21UR-synth is specifically detected in transgenic strains and is susceptible to β-elimination only in the henn-1(tm4477) background. Arrowhead represents migration of a 21 nt size marker. 21UR-synth blot was reprobed for miR-1. Endogenous ♀21UR-2502 is shown as a control. (C–E) 21UR-synth is a prg-1-dependent, germline-enriched 21U RNA. 21UR-synth detection by Taqman RT-qPCR (C) and northern blot (D,E) is greatly decreased in prg-1(tm872) and glp-4(bn2) germline-deficient mutant animals, but intact in rde-4(ne301) mutant animals. Error bars: ±1 SD of three biological replicates. (F) anti-PRG-1 antibody immunopurifies PRG-1 complexes. CL: crude lysate, RIP: RNA immunoprecipitation. (G) 21UR-synth is bound by endogenous PRG-1. Error bars: ±1 SD of two technical replicates; data are representative of two independent experiments. (H,I) Loss of the core motif dramatically decreases 21UR-synth expression by northern blot (H) and Taqman qRT-PCR (I). Error bars: ±1 SD of three biological replicates.

Figure 4. A 5′ cytidine in the core upstream motif promotes male germline expression pattern of 21UR-synth.

(A) Schematic of transgenes with 5′ nt of core motif mutated. (B) Left: Endogenous ♂21UR-1258 and ♀21UR-2502 peak during spermatogenesis (sp.) and oogenesis (oo.), respectively. Right: Germline enrichment patterns are recapitulated in him-8(e1489) male and fem-1(hc17) female animals. Error bars: ±1 SD of three biological replicates. (C) The male expression pattern of 21UR-synth from ♂Tg1258 is disrupted by core motif mutation in ♂C>A1258. Error bars: ±1 SD of three biological replicates. (D) The female expression pattern of 21UR-synth from ♀Tg2502 is disrupted by core motif mutation in ♀A>C2502, but expression in fem-1(hc17) female is not lost. Error bars: ±1 SD of three biological replicates. (E) Mutating the 5′ nt of the core motif does not affect 21UR-synth prg-1 dependence.

Figure 5. 21U RNA sequences are specified by the genomic positions of upstream core motifs.

(A) Schematic of transgenes with 5′ nt of 21U RNA mutated. (B–C) Mutation of the 5′ genomic thymidine disrupts expression of 21UR-synth by northern blot (B) and Taqman assay (C). (D) 21U RNA abundances correlate with distances downstream of core motifs. Miniclustered 21U RNAs with 37–40 nt spacer lengths are more abundant than solitary 21U RNAs. Asterisks indicate Welch's t-tests, p<0.05. Error bars: ±1 SEM. (E) Optimal downstream windows are more thymidine-rich for shared core motifs than non-shared (Welch's t-test, p = 2.5e-46). The number of genomic thymidines located 35–42 nt downstream of each GTTTC-containing motif was counted. (F) 21U RNA miniclusters are significantly biased for being composed of 21U RNAs with the same, as opposed to opposite, germline enrichment than expected if the same 21U RNAs were randomly paired.

Figure 6. 21U RNAs represent independent transcriptional units.

(A–B) 21UR-synth expressed from a minimal transcriptional cassette shows prg-1 dependence, rde-4 independence, and germline enrichment by northern blot (A) and Taqman assay (B). (C) The male expression pattern of 21UR-synth from ♂Min1415 is disrupted by core motif mutation in ♂MinC>A1415. Error bars: ±1 SD of three biological replicates. (D) Schematic of transgenes encoding two closely adjacent 21U RNAs. (E) Scrambling the core motif upstream of 21UR-synth abrogates 21UR-synth, but not 21UR-synthB, expression levels. (F) The ♂Tg1258, ♂C>A1258, ♀Tg2502, and ♀A>C2502 transgenes, but not the ♂Scram1258 or ♀Scram2502 transgenes, express from single copy insertions on chromosome.